HindIII liposomes suppress delayed-type hypersensitivity responses in vivo and induce epidermal IL-10 in vitro

The purpose of this study was to evaluate the effects of resident islet macrophage activation on beta cell function. Treatment of freshly isolated rat islets with TNF-alpha and LPS results in a potent inhibition of glucose-stimulated insulin secretion. The inhibitory actions of TNF + LPS are mediated by the intraislet production and release of IL-1 followed by IL-1-induced inducible nitric oxide synthase (iNOS) expression by beta cells. The IL-1R antagonist protein completely prevents TNF + LPS-induced nitrite production, iNOS expression and the inhibitory effects on glucose-stimulated insulin secretion by rat islets. Resident macrophages appear to be the source of IL-1, as a 7-day culture of rat islets at 24 degrees C (conditions known to deplete islets of lymphoid cells) prevents TNF + LPS-induced iNOS expression, nitrite production, and the inhibitory effects on insulin secretion. In addition, macrophage depletion also inhibits TNF + LPS-induced IL-1alpha and IL-1beta mRNA expression in rat islets. Immunocytochemical colocalization of IL-1beta with the macrophage-specific marker ED1 was used to provide direct support for resident macrophages as the islet cellular source of IL-1. IL-1beta appears to mediate the inhibitory actions of TNF + LPS on beta cell function as TNF + LPS-induced expression of IL-1beta is fourfold higher than IL-1alpha, and Ab neutralization of IL-1beta prevents TNF + LPS-induced nitrite production by rat islets. These findings support a mechanism by which the activation of resident islet macrophages and the intraislet release of IL-1 may mediate the initial dysfunction and destruction of beta cells during the development of autoimmune diabetes.     

Ciliary neurotrophic factor potentiates the beta-cell inhibitory effect of IL-1beta in rat pancreatic islets associated with increased nitric oxide synthesis and increased expression of inducible nitric oxide synthase

Proinflammatory cytokines are implicated as effector molecules in the pathogenesis of IDDM. Interleukin-6 (IL-6) alone or in combination with IL-1beta inhibits glucose-stimulated insulin release from isolated rat pancreatic islets by unknown mechanisms. Here we investigated 1) if the effects of IL-6 are mimicked by ciliary neurotrophic factor (CNTF), another member of the IL-6 family of cytokines signaling via gp130, 2) the possible cellular mechanisms for these effects, and 3) if islet endocrine cells are a source of CNTF. CNTF (20 ng/ml) potentiated IL-1beta-mediated (5-150 pg/ml) nitric oxide (NO) synthesis from neonatal Wistar rat islets by 31-116%, inhibition of accumulated insulin release by 34-49%, and inhibition insulin response to a 2-h glucose challenge by 31-36%. CNTF potentiated IL-1beta-mediated NO synthesis from RIN-5AH cells by 83%, and IL-1beta induced islet inducible NO-synthase (iNOS) mRNA expression fourfold. IL-6 (10 ng/ml) also potentiated IL-1beta-mediated NO synthesis and inhibition of insulin release, whereas beta-nerve growth factor (NGF) (5 or 50 ng/ml) had no effect. mRNA for CNTF was expressed in rat islets and in islet cell lines. In conclusion, CNTF is constitutively expressed in pancreatic beta-cells and potentiates the beta-cell inhibitory effect of IL-1beta in association with increased iNOS expression and NO synthesis, an effect shared by IL-6 but not by beta-NGF. These findings indicate that signaling via gp130 influences islet NO synthesis associated with iNOS expression. We hypothesize that CNTF released from destroyed beta-cells during the inflammatory islet lesion leading to IDDM may potentiate IL-1beta action on the beta-cells.     

Double-stranded RNA-induced inducible nitric-oxide synthase expression and interleukin-1 release by murine macrophages requires NF-kappaB activation

The effects of double-stranded RNA (synthetic polyinosinic-polycytidylic acid; poly(I-C)) on macrophage expression of inducible nitric-oxide synthase (iNOS), production of nitric oxide, and release of interleukin-1 (IL-1) were investigated. Individually, poly(I-C), interferon-gamma (IFN-gamma), and lipopolysaccharide (LPS) stimulate nitrite production and iNOS expression by RAW 264.7 cells. In combination, the effects of poly(I-C) + IFN-gamma are additive, while poly(I-C) does not further potentiate LPS-induced nitrite production. These results suggest that poly(I-C) and LPS may stimulate iNOS expression by similar signaling pathways, which may be independent of pathways activated by IFN-gamma. LPS-induced iNOS expression is associated with the activation of NF-kappaB. We show that inhibition of NF-kappaB by pyrrolidinedithiocarbamate prevents poly(I-C) + IFN-gamma-, poly(I-C) + LPS-, and LPS-induced iNOS expression, nitrite production and IkappaB degradation by RAW 264.7 cells. The effects of poly(I-C) on iNOS expression appear to be cell-type specific. Poly(I-C), alone or in combination with IFN-gamma, does not stimulate, nor does poly(I-C) potentiate, IL-1-induced nitrite production by rat insulinoma RINm5F cells. In addition, we show that the combination of poly(I-C) + IFN-gamma stimulates iNOS expression, nitrite production, IkappaB degradation, and the release of IL-1 by primary mouse macrophages, and these effects are prevented by pyrrolidinedithiocarbamate. These findings indicate that double-stranded RNA, in the presence of IFN-gamma, is a potent activator of macrophages, stimulating iNOS expression, nitrite production, and IL-1 release by a mechanism which requires the activation of NF-kappaB.     

Inhibitory effects of streptozotocin, tumor necrosis factor-alpha, and interleukin-1beta on glucokinase activity in pancreatic islets and gene expression of GLUT2 and glucokinase

Treatment of streptozotocin (ST), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) resulted in destroying insulin-secreting beta-cells of pancreatic islets and impairment of islet glucose oxidation and glucose-induced insulin secretion. IL-1beta and TNF-alpha inhibited insulin release and glucose utilization and oxidation. It was shown that the inhibitory effects of ST, IL-1beta, and TNF-alpha were due to impaired glucokinase activity. Glucokinase activity was severely impaired by ST, IL-1beta, and TNF-alpha treatments, as confirmed by assaying enzymes and nucleotides associated with glycolysis and glucose oxidation. On the other hand, nitric oxide was a factor of the deleterious effects of IL-1beta, TNF-alpha, and ST on pancreatic islets. Incubation of mouse pancreatic islets with ST at various concentrations of impairing insulin secretion resulted in generation of nitrite, stimulation of islet guanylyl cyclase and accumulation of cGMP, and inhibition of pancreatic islet mitochondrial aconitase activity to degree similar to those raised by IL-1beta and TNF-alpha. When the effects of IL-1beta and TNF-alpha on the gene expression of pancreatic GLUT2 and glucokinase were examined, the level of GLUT2 and glucokinase mRNA in pancreatic islets was significantly decreased. This suggested that IL-1beta and TNF-alpha downregulate gene expression of GLUT2 and glucokinase in pancreatic beta-cells.     

An increase in intracellular cyclic AMP modulates nitric oxide production in IFN-gamma-treated macrophages

Macrophages treated with IFN-gamma alone are stimulated to produce nitric oxide. The level of nitric oxide production can be enhanced significantly when IFN-gamma treatment is combined with other agents (e.g., LPS, TNF-alpha, IL-2, etc.). We tested the hypothesis that cAMP plays a role in the IFN-gamma-induced activation of macrophages. Our experiments indicate that factors that increase the concentration of cAMP in the murine macrophage cell line ANA-1 can also enhance IFN-gamma-induced production of nitric oxide. PGE2 and cholera toxin increased the production of nitrite (an indicator of nitric oxide production) in IFN-gamma-treated ANA-1 macrophages by at least twofold. These factors produced no increase in nitric oxide production in the absence of IFN-gamma treatment. The increase in nitric oxide production corresponded to an increase in the accumulation of nitric oxide synthase mRNA without a change in stability of mRNA. Dibutyryl cAMP and Sp-cAMPs (a selective activator of cAMP-dependent protein kinase I and II) also increased nitric oxide production in IFN-gamma-treated macrophages. However, at very high concentrations (i.e., >100 microM), the stimulatory effect was decreased. These studies indicate that elevation of intracellular cAMP causes a dose-dependent, biphasic alteration of IFN-gamma-induced nitric oxide production in murine macrophages. Moreover, they suggest that agents that affect nitric oxide synthesis may do so via modulation of the cAMP second messenger system.     

Acid sphingomyelinase-derived ceramide is not required for inflammatory cytokine signalling in murine macrophages

Sphingomyelin hydrolysis is induced in myeloid cell-lines by tumour necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1beta), and interferon gamma (IFN-gamma). Ceramide, a product of sphingomyelin hydrolysis, recapitulates many of the cellular responses elicited by these cytokines, and this has lead to the hypothesis that ceramide is a second messenger of cytokine signalling. Sphingomyelin hydrolysis is catalysed by an acid spingomyelinase (ASMase) and one or more neutral sphingomyelinases (NSMase); both ASMase and NSMase are activated during cytokine signalling. In the present study, the contribution of ASMase to TNF-alpha, IL-1beta, and IFN-gamma signalling in murine macrophages was addressed. Cytokine-induced responses were compared in macrophages derived from the bone marrow of AMSase null and wild-type mice. Specifically, TNF-alpha-and IFN-gamma-induced nitric oxide production and TNF-alpha- and IL-1beta-induced expression of the alpha-chemokine, KC, were intact in ASMase null macrophages. Furthermore, TNF-alpha induction of p42/p44 ERK and p38-MAPK phosphorylation, c-jun kinase activation, and IkappaBalpha degradation were normal. Also normal in ASMase null macrophages was TNF-alpha-, IL-1beta- and IFN-gamma-induced expression of a panel of early response genes. It is concluded that ASMase is non-essential for the inflammatory signals activated in murine macrophages by TNF-alpha, IL-1beta and IFN-gamma.     

Generation of nitric oxide and induction of major histocompatibility complex class II antigen in macrophages from mice lacking the interferon gamma receptor

Availability of mice with a targeted disruption of the interferon gamma (IFN-gamma) receptor gene (IFN-gamma R0/0 mice) made it possible to examine parameters of macrophage activation in the absence of a functional IFN-gamma receptor. We asked to what extent other cytokines could replace IFN-gamma in the induction of nitric oxide or major histocompatibility complex class II antigen (Ia) expression in peritoneal macrophages. In thioglycollate-elicited macrophages from wild-type mice, tumor necrosis factor (TNF) alone was virtually ineffective in inducing release of NO2- (the endproduct of nitric oxide generation), but TNF enhanced NO2- release in the presence of IFN-gamma. In macrophages from IFN-gamma R0/0 mice, which were unresponsive to IFN-gamma, TNF completely failed to stimulate NO2- release. The stimulatory actions of IFN-alpha/beta on NO2- release were indistinguishable in wild-type and IFN-gamma R0/0 macrophages: IFN-alpha/beta was ineffective on its own, showed marginal stimulation of NO2- release in combination with TNF, and was moderately effective in the presence of lipopolysaccharide. The level of constitutive Ia antigen expression was not significantly different in peritoneal macrophages from wild-type and IFN-gamma R0/0 mice. An increased Ia expression was induced by IL-4 and granulocyte-macrophage colony-stimulating factor in both wild-type and IFN-gamma R0/0 macrophages, but the magnitude of this induction was less than with optimal concentrations of IFN-gamma in macrophages from wild-type mice. IFN-alpha/beta showed only a minor stimulatory effect on Ia expression in both wild-type and IFN-gamma R0/0 macrophages. Simultaneous treatment of wild-type macrophages with IFN-alpha/beta and IFN-gamma reduced the IFN-gamma-induced Ia expression in wild-type macrophages, but IFN-alpha/beta did not show an inhibitory effect on IL-4- or granulocyte-macrophage-colony-stimulating factor-induced Ia expression in either wild-type or IFN-gamma R0/0 macrophages. The important role of IFN-gamma in the regulation of the induced expression of major histocompatibility complex class II antigen was confirmed by showing that after systemic infection with the BCG strain of Mycobacterium bovis resident peritoneal macrophages from IFN-gamma R0/0 mice had a lower level of Ia expression than macrophages from wild-type mice. The inability of other cytokines to substitute fully for IFN-gamma in macrophage activation helps to explain the earlier observed decreased resistance of IFN-gamma R0/0 mice to some infections.     

Mechanism of suppression of macrophage nitric oxide release by IL-13: influence of the macrophage population

IL-13 is a cytokine produced by T lymphocytes, mast cells, basophils, and certain B cell lines that up-regulates or inhibits various macrophage functions. In the present study we analyzed the mechanisms of suppression of nitric oxide (NO) release by IL-13 in the macrophage cell line J774A.1 and in thioglycolate-elicited mouse peritoneal macrophages. In both cell types efficient reduction (>80%) of NO production required treatment of the macrophages with IL-13 for at least 7 h before stimulation with IFN-gamma and LPS. In J774A.1 cells, increasing concentrations of IFN-gamma partially antagonized the suppression mediated by IL-13, whereas in peritoneal macrophages, the inhibitory effect of IL-13 was largely independent of the concentrations of IFN-gamma and LPS. In J774A.1 cells, IL-13 strongly reduced both the mRNA and protein levels of inducible nitric oxide synthase (iNOS, NOS-2), as determined by Northern blot analysis and immunoprecipitation. In peritoneal macrophages, in contrast, IL-13 decreased iNOS protein and enzyme activities after 8 to 48 h of stimulation, without altering the expression of iNOS mRNA. Pulse labeling with [35S]methionine revealed that IL-13 caused a 4.7-fold reduction of the de novo synthesis of iNOS protein in these cells. These data demonstrate for the first time that IL-13 is capable of regulating iNOS at both the mRNA and translational levels and underline the important influence of the macrophage population when studying mechanisms of cytokine functions.     

Role of iron in the potentiation of anthracycline cardiotoxicity: identification of heart cell mitochondria as a major site of iron-anthracycline interaction

The effect of nitric oxide on the lipopolysaccharide (LPS)-induced cytokine production by alveolar macrophages was studied. When alveolar macrophages were cultured, substantial amounts of interleukin-1(IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha(TNF-alpha), and nitric oxide are produced upon stimulation with LPS. Inhibition of the nitric oxide production by the L-arginine analogue N(G)-monomethyl-L-arginine (NMMA), resulted in an increase of IL-1(beta) and IL-6, whereas the TNF-alpha concentrations remained unchanged, suggesting specific inhibitory effects of nitric oxide on the LPS-stimulated cytokine production by alveolar macrophages. The observed cytokine-modulation properties of nitric oxide did not result from cytotoxic actions of the oxidation of L-arginine on macrophages, since nitric oxide synthesis did not affect the viability of the alveolar macrophages. Conversely the nitric oxide donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) induced dose-dependent inhibition of IL-1 production in LPS-stimulated alveolar macrophages in which endogenous nitric oxide production was blocked. The results indicate that nitric oxide can affect the LPS-induced IL-1beta and IL-6 secretion by alveolar macrophages in an autoregulatory way and are discussed in view of the important physiologic consequences this autoregulation by nitric acid oxide may have.     

Dexamethasone prevents interleukin-1beta-mediated inhibition of rat islet insulin secretion without decreasing nitric oxide production

The deleterious effects of interleukin 1 (IL-1) on insulin-producing beta-cells are partly mediated by the generation of the free radical nitric oxide (NO). We aimed to assess the effect of several steroidal hormones on IL-1beta-induced inhibition of rat islet insulin secretion in vitro, and their possible regulatory effects on NO production. Incubation of newborn rat islets for 24 h in the presence of 150 pg/ml IL-1beta revealed that dexamethasone dose-dependently attenuated the inhibitory effect of IL-1beta on insulin release in response to a 2-h glucose challenge. Physiological and supraphysiological concentrations of testosterone, 17beta-estradiol, progesterone, 1,25-dihydroxyvitamin-D3 and vitamin D analogues (KH1060 and MC1288) were ineffective. Dexamethasone (1 microM) increased the production of NO in IL-1beta-treated rat islets, as measured by the concentration of nitrite in the media. However, 1-5 microM dexamethasone inhibited IL-1beta-induced NO production by RIN cells. Dexamethasone (1 microM) did not affect the inhibitory action of the NO donor S-nitroso penicillamine (500 microM) on rat islet insulin secretion. We conclude that dexamethasone partially protects against IL-1beta-induced inhibition of rat islet insulin secretion, an effect which is not mediated through modulation of the NO pathway.     

Regulation of inducible NO synthase expression by endothelin in primary cultured glial cells

Nitric oxide (NO), initially identified as an endothelium-derived relaxing factor, is a molecular mediator that has been implicated in many physiological and pathological processes. In primary cultured rat glial cells, a combination of inflammatory cytokines (tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta)) and bacterial lipopolysaccharide (LPS) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) markedly inhibited TNF-alpha/IL-1beta-induced and LPS-induced nitrite production and iNOS expression, although ET by itself had no effect. The inhibitory effect of ETs appears to be mediated by ET(B) receptors. Forskolin also inhibited the iNOS expression. By contrast, pretreatment with ET for 24 hours enhanced LPS-induced nitrite production and iNOS expression. This stimulatory effect of ETs was suppressed by calphostin C, a protein kinase C inhibitor, and pretreatment with phorbol ester enhanced LPS-induced iNOS expression. Our findings present the possibility that ET has dual effects on iNOS expression in glial cells.     

Pathophysiological role of nitric oxide in rat experimental colitis

Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to the pathophysiology of ulcerative colitis. A 2,4,6-trinitrobenzenesulfonic acid sodium salt (TNBS) colitis model was established to examine the effect of selective iNOS inhibition, by S-(2-aminoethyl) isothiouronium bromide (ITU), on colonic mucosal cell damage and inflammation. Rats, killed 7 days after TNBS, had increased colonic mucosal levels of iNOS and interleukin-8 (IL-8), in addition to severe colonic inflammation which was characterized by significantly increased colon weight, damage score and colonic myeloperoxidase activity (MPO) (a marker of neutrophil influx). TNBS-treated rats had markedly decreased body weight and thymus weight. Administration of colitic rats with ITU significantly inhibited iNOS activity/expression and tended to reduce mucosal levels of IL-8, but no effect on MPO activity was observed. Following ITU therapy, colitic rats had reduced colonic damage and losses in body weight and thymus weight were reversed. Improvement of TNBS colitis by ITU suggested that excess NO, produced by iNOS, may have contributed to the initiation/amplification of colonic disease, by mechanisms including enhancement of IL-8 release. NO-mediated enhancement of pro-inflammatory cytokine release was further investigated in vitro. Lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) stimulated release of nitrite, lactate dehydrogenase (LDH), TNF alpha, IL-1 beta and IL-8 from rat peritoneal macrophages, all of which were significantly reduced by ITU. This suggests that NO-mediated cell damage enhances pro-inflammatory mediator release from macrophages. In addition, enhancement of IL-8 and TNF alpha release was also partially NO-dependent in activated peritoneal neutrophils. Therefore, the amelioration of TNBS colitis by ITU could include inhibition of NO-mediated pro-inflammatory cytokine release.     

Transcriptional basis for hyporesponsiveness of the human inducible nitric oxide synthase gene to lipopolysaccharide/interferon-gamma

The work reported here resolves, at the level of gene regulation, the controversy as to whether or not human monocytes/macrophages can produce nitric oxide (NO) when stimulated with lipopolysaccharide (LPS), with or without co-stimulation by interferon-gamma (IFN-gamma). Studies included structural comparison of the promoters for human and mouse inducible NO synthase (iNOS) genes, transfection and assay of human and mouse iNOS promoter regions in response to LPS +/- IFN-gamma, and electrophoretic mobility shift assays of kappa B response elements. Two explanations for hyporesponsiveness of the human iNOS promoter to LPS +/- IFN-gamma were found: (1) multiple inactivating nucleotide substitutions in the human counterpart of the enhancer element that has been shown to regulate LPS/IFN-gamma induced expression of the mouse iNOS gene; and (2) and absence of one or more nuclear factors in human macrophages (e.g., an LPS-inducible nuclear factor-kappa B/Rel complex), that is (are) required for maximal expression of the gene. The importance of resolution of this controversy is that future research in this area should be directed toward the understanding of alternative mechanisms that can result in the successful production of NO.